This project will investigate molecular mechanisms of deletion mutagenesis in human cells. Attention is focused on comparing the quantitative yield and qualitative nature of mutations arising spontaneously or induced by radiation, in closely related cell lines that vary in p53 status. Some evidence suggests that all p53 mutations are not identical in their phenotypic effects. In fact, it is unclear whether increased mutability in p53 mutants is due to the absence of functional protein or the presence in higher concentration of a mutant form that retains certain activity. DNA deletions may arise by recombination between homologous regions. An hprt has been developed with an extra, inactivating exon. Once it is integrated into a cell, deletion of the extra exon leads to mRNA coding for a functional protein, and such cells can be selected easily. Since cloning sites flanking the extra exon allow sequences of interest to be inserted, the involvement of homology in deletion mutagenesis in p53 wild-type and mutant cells can be explored systematically.
Specific aim 1 will determine whether p53 acts on mutation act in a positive or negative fashion. The hypothesis that various p53 mutations exhibit different quantitative effects on mutagenesis will be tested. First, a p53 null line will be constructed, using a promoterless-neo approach. Second, to see if there is a negative effect of p53 on mutation, i.e., that lack of p53 functional protein leads to increased mutability in the p53 null cells, spontaneous mutation rate will be determined, as will mutation, wild-type and dominant-negative or gain-of function p53 mutant alleles will be transfected back to the null cell. These lines will be used to conduct comparative quantitative mutagenesis studies.
Specific aim 2 will use the minigene system to study effects of sequence homology on deletion mutagenesis. First, vectors will be constructed with direct or inverted Alu repeats, with various degrees of homology. These will be transfected into p53 wild-type and mutant cells. Second, mutagenesis studies will determine the spontaneous rate of deletion/recombination, and the induced frequencies after exposure to ultraviolet light or X-rays. The molecular structure of recombinants will be analyzed to see if there is less stringency for deletions occurring between homologous sequences in the p53 mutant line. Finally, cell lines from aim 1 will be examined with the minigene system.
